Synthesis and anticancer evaluation of bis(benzimidazoles), bis(benzoxazoles), and benzothiazoles

Article abstract of DOI:10.1016/j.bmc.2006.05.007

Four classes of UK-1 analogues were synthesized and their cytotoxicity testing against human A-549, BFTC-905, RD, MES-SA, and HeLa carcinoma cell lines was determined. The results revealed that UK-1 and four of these analogues (15-18) are potent against the cancer cell lines. In particular, compound 16 is more potent than UK-1 against A-549 and HeLa cell lines, and compounds 15, 17, and 18 selectively exhibit potent cytotoxic activity against the BFTV-905 cells (IC₅₀ 9.6 μM), A-549 cells (IC₅₀ 6.6 μM), and MES-SA cells (IC₅₀ 9.2 μM), respectively.

Full text of DOI:10.1016/j.bmc.2006.05.007

Bioorganic & Medicinal Chemistry 14 (2006) 6106–6119
Synthesis and anticancer evaluation of bis(benzimidazoles),
bis(benzoxazoles), and benzothiazoles
Shu-Ting Huang,a I-Jen Hseib and Chinpiao Chena,*
aDepartment of Chemistry, National Dong Hwa University, Shoufeng, Hualien 974, Taiwan
bDepartment of Medical Laboratory Science and Biotechnology, College of Medicine, Tzu Chi University, Hualien 970, Taiwan
Received 10 March 2006; revised 1 May 2006; accepted 2 May 2006
Available online 22 May 2006
Abstract—Four classes of UK-1 analogues were synthesized and their cytotoxicity testing against human A-549, BFTC-905, RD,
MES-SA, and HeLa carcinoma cell lines was determined. The results revealed that UK-1 and four of these analogues (15–18)
are potent against the cancer cell lines. In particular, compound 16 is more potent than UK-1 against A-549 and HeLa cell lines,
and compounds 15, 17, and 18 selectively exhibit potent cytotoxic activity against the BFTV-905 cells (IC50 9.6 lM), A-549 cells
(IC50 6.6 lM), and MES-SA cells (IC50 9.2 lM), respectively.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
DNA in a magnesium ion-dependent fashion, UK-1
inhibits the activity of human topoisomerase II.
Taniguchi et al. isolated the bis(benzoxazole) natural
product UK-1 from the mycelial cake of an actinomy-
cete strain 517-02.1–3 Subsequently, Tsuji et al. isolat-
ed AJI9561 from the mycelium extract of
Streptomyces sp. AJ9561.4 UK-1 exhibits significant
cytotoxic activity against B16, HeLa, and P338 cells,
but does not exhibit any growth-inhibiting activity
against Gram-positive bacteria, yeasts or fungi at up
to 100 lg/mL.1 AJI9561 exhibited cytotoxic activity
against both Jurkat and P388 cells with IC50 values
of 0.88 and 1.63 lM, respectively. However, the meth-
yl derivative MUK-1 and alkaline hydrolysate demeth-
yl derivative DMUK-1 both exhibit activity against
Gram-positive and Gram-negative bacteria.2,3 MUK-
1 is also active against yeast and filamentous fungi.2
Kerwin et al. investigated the metal ion coordination
by UK-1 and found that UK-1 binds Zn2+ and
Mg2+ to form stable complexes with an overall stoi-
chiometry of 1:1.5 Fe3+ ions are bound slightly less
strongly by UK-1, and Ca2+ ions form weak complex-
es with this ligand. In the presence of Mg2+, UK-1
binds to double-stranded DNA ten times more tightly
than in the absence of Mg2+. Like the antitumor agent
quinobenzoxazines, which also bind Mg2+ and bind to
O
O
OR1
R2O
OH
O
N
O
N
HO
N
O
N
O
Me
UK-1: R1 = Me, R2 = H
MUK-1: R1 = Me, R2 = Me
DMUK-1: R1 = H, R2 = H
AJI9561
Kerwin et al. subsequently reported the activity of UK-1
against a wide range of human cancer cell lines.6 UK-1
exhibits a wide spectrum of potent anticancer activity
against leukemia, lymphoma, and certain solid tumor-
derived cell lines, with IC50 values of as low as 20 nM,
but is inactive against Staphylococcus aureus. A series
of analogues of the bis(benzoxazole) natural product
UK-1 in which the carbomethoxy-substituted benzoxaz-
ole ring of the natural product was modified were pre-
pared, and their anticancer and antibacterial properties
evaluated. An analogue of UK-1, in which the carbome-
thoxy-substituted benzoxazole ring was replaced by a
carbomethoxy-substituted benzimidazole ring (com-
pound B), was inactive against human cancer cell lines
and the two strains of S. aureus. In contrast, a simplified
analogue in which the carbomethoxy-substituted benz-
oxazole ring was replaced with a carbomethoxy group
Keywords: Bis(benzoxazole); Bis(benzoxazole); Benzothiazole; Anti-
cancer, Cytotoxicity.
*
Corresponding author. Tel.: +886 3 8633597; fax: +886 3 8630475;
e-mail: chinpiao@mail.ndhu.edu.tw
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.007

S.-T. Huang et al. / Bioorg. Med. Chem. 14 (2006) 6106–6119
6107
(compound A) was almost as active as UK-1 against the
four cancer cell lines examined but exhibited no activity
against S. aureus. The benzylated UK-1 analogue (com-
pound C) is more active than UK-1 against HT-29 cells,
about 10-fold less potent than UK-1 against MCF-7 and
PC-3 cells, and 20-fold less active than UK-1 against
HL60 cells. Metal ion binding investigations of these
analogues indicate that they bind both Zn2+ and Ca2+
approximately as strongly as UK-1. The non-cytotoxic
benzimidazole UK-1 analogue binds Mg2+ ions 50 times
more weakly than UK-1, while the simple benzoxazole
analogue binds Mg2+ ions almost as effectively as UK-
1. These results are evidence of the involvement of
Mg2+ ion binding in the selective cytotoxicity of UK-1
and its provision of a minimal pharmacophore for the
selective cytotoxic activity of the natural product.
dates the natural product’s selective activity against can-
cer cells.
2. Chemistry
A number of analogues of UK-1 in which the carbome-
thoxy-substituted benzoxazole ring was either modified
or deleted were prepared. These UK-1 analogues were
synthesized in a manner that was analogous to the pre-
viously reported total synthesis of the natural product6–8
with some improvements. An analogue of UK-1, in
which the carbomethoxy-substituted benzoxazole ring
is replaced with a substituted benzimidazole ring, was
prepared according to the Schemes 1–3. Compound 6
was obtained in five steps, starting with the esterification
of 2-aminobenzoic acid (1) with diazomethane to yield
methyl 2-aminobenzoate (2) in 94% yield (Scheme1).
The nitration of compound 2 gave 2-acetylamino-3-ni-
tro-benzoic acid methyl ester 3 with a 37% yield, and
2-acetylamino-5-nitro-benzoic acid methyl ester 4 with
a 61% yield.9 Following separation, the acetyl group
of 3 was cleaved using sodium methoxide10 and treated
with diazomethane to esterify partially hydrolyzed ester
5 with a 87% yield. The nitro group of 5 was reduced by
hydrogenation on Pd/C to yield 2,3-diaminobenzoic acid
methyl ester 6 with a 96% yield.
CO2Me
CO2Me
CO2Me
O
N
O
N
HN
N
HO
Bn
HO
OH
N
N
O
O
C
A
B
Smith et al. synthesized three new analogues of UK-1
and evaluated their efficacies as topoisomerase II inhib-
itors.7 Their results revealed that UK-1 and two of these
analogues (compounds D and E) are catalytic inhibitors
of topoisomerase II, whereas compound F shows no
measurable activity, and identify a critical structural
motif required for enzyme inhibition.
Compound 12 was prepared in five steps, starting with
the esterification of salicylic acid 7 by diazomethane to
generate methyl salicylate 8 in quantitative yield
(Scheme 2). The hydroxyl group of 8 was protected with
benzyl ether by treating with benzyl bromide and potas-
sium carbonate to give 9 in 93% yield.11 Compound 9
was transformed to the carboxylic acid 10 in quantita-
tive yield by saponification reaction. Compound 10
was coupled with 6 by treating it with dicyclohexylcar-
bodiimide to furnish amide 11 with a 83% yield which
was then converted to benzimidazole 12 in 87% yield
via cyclization-dehydration by refluxing in acetic acid.12
Compound 12 was saponified by 5 M NaOH to give 13
in 97% yield. The benzyl groups in 12 and 13 were
cleaved by hydrogenation to generate the corresponding
phenols 14 and 15 in 97% and 60% yields, respectively.
CO2Me
O
N
O
N
O
N
HO
N
N
O
N
O
O
D
E
F
A number of structural analogues of this natural prod-
uct were prepared to understand the structural basis of
the selective cytotoxicity of UK-1. A comparison of
the anticancer activity of these compounds with that
of their structurally simplified analogues of UK-1 eluci-
Compound 13 was transformed to acyl chloride by
treatment with thionyl chloride and then coupled with
CO2CH3
NHAc
CO2CH3
NHAc
CO2H
NH2
CO2CH3
NH2
Ac2O, rt, 1 h;
conc HNO3/Ac2O/AcOH,
5-15oC, 2.5 h
CH2N2
ether
+
NO2
O2N
4
3
1
2
NaOMe, MeOH
CH2N2, ether
CO2CH3
NH2
CO2CH3
NH2
H2, Pd/C, MeOH
NH2
NO2
6
5
Scheme 1. Synthesis of 2,3-diaminobenzoic acid methyl ester 6.

6108
S.-T. Huang et al. / Bioorg. Med. Chem. 14 (2006) 6106–6119
CO2H
OBn
CO2CH3
CO2CH3
CO2H
OH
BnBr, K2CO3,
1.4N NaOH,
OH
OBn
CH2N2
ether
dioxane,
reflux, 1 h
CH2Cl2, MeOH,
reflux, 9 h
8
10
9
7
6, DCC,
DMAP, CH2Cl2
H2N
OBn
HN
CO2CH3
CO2CH3
CO2H
OBn
N
OBn
5N NaOH,
THF
N
AcOH,
120-130oC
N
H
N
H
O
11
12
13
H2/Pd-C,
MeOH
H2/Pd-C,
MeOH
CO2CH3
OH
CO2H
OH
N
N
N
H
N
H
14
15
Scheme 2. Synthesis of benzimidazole analogues 12–15.
CO2CH3
BnO
BnO
NH2
O
SOCl2, reflux; 6,
AcOH,
HN
CO2CH3
N
NH
N
NH
OBn
120-130oC
pyridine, CH2Cl2
N
N
HO2C
N
H
N
H
16
17
13
H2/Pd-C,
MeOH
5N NaOH,
THF
HO
BnO
CO2CH3
N
NH
CO2H
N
NH
N
N
N
H
N
H
19
18
Scheme 3. Synthesis of bis(benzimidazole) analogues 17–19.
6 to give 16 in 56% yield (Scheme 3). Compound 16 was
refluxed in acetic acid to form the bis(benzimidazole) 17
in 85% yield, and this 17 was converted to 18 in quanti-
tative yield by hydrogenation, and to 19 in 66% yield by
saponification.
a cyclization-dehydration product 23 in 86% yield,
which was saponified to give 24 in 84% yield. Com-
pound 24 was converted to acyl chloride by treating it
with oxalyl chloride and N,N-dimethylformamide, and
then reacting it with 20 to give 26 in 76% yield. Com-
pound 23 was refluxed with p-toluenesulfonic acid in tol-
uene to give compound 25 in 51% yield. Final
cyclodehydration and debenzylation were performed
by refluxing 26 with p-toluenesulfonic acid in p-xylene
to give the UK-1 in 43% yield.
UK-1 is typically synthesized following the procedure of
Kerwin et al. (Scheme 4).5 Compound 20 was prepared
from 2-amino-3-hydroxybenzoic acid by treating with
diazomethane. Compound 10 was transformed to acyl
chloride by treating with oxalyl chloride and N,N-di-
methylformamide, and then reacted with 20 to give the
amide 22 in 96% yield. Heating 22 to 230 ꢁC produced
One of the UK-1 analogues, 35, was prepared from
salicylic acid presented in Scheme 5. Salicylic acid

S.-T. Huang et al. / Bioorg. Med. Chem. 14 (2006) 6106–6119
6109
CO2Me
N
HO
O
25
CO2H
NH2
CO2Me
NH2
p-TsOH,
toluene, reflux
CH2N2
ether
HO
CO2Me
O
OH
pyridine,
CH2Cl2
230oC
OH
O
HN
20
N
OBn
23
CO2H
OBn
OBn
COCl
(COCl) ,
CO2Me
2
OBn
DMF
CH2Cl2
22
5N NaOH,
THF, reflux
10
21
HO2C
H3CO2C
OH
1.(COCl)2, DMF,
CH2Cl2
CO2CH3
O
HO
p-TsOH,
N
O
N
O
HN
p-xylene,
reflux
2. 20, pyridine,
CH2Cl2
OBn
BnO
N
N
O
O
24
26
UK-1
Scheme 4. Synthesis of UK-1.
CO2H
CO2H
CO2CH3
OCH3
OCH3
6N NaOH
MeOH
MeI, K2CO3
acetone
OH
1. (COCl)2, DMF, CH2Cl2
28
2.
pyridine
CH2Cl2
CO2CH3
OH
27
7
NH2
29
CO2CH3
CO2CH3
OH
OCH3
OCH3
OH
p-TsOH
p-xylene
H
N
O
O
CO2CH3
N
N
O
32
31
30
5N NaOH
THF
H3CO2C
HO
H3CO2C
OCH3
HO2C
H3CO
1. (COCl)2,
DMF, CH2Cl2
p-TsOH
p-xylene
O
N
O
N
HN
O
OCH3
2. 29, pyridine
CH2Cl2
O
O
N
N
33
34
35
Scheme 5. Synthesis of 31–35.
was transformed to 27 by treating it with iodome-
thane and potassium carbonate.13 It was then treated
with sodium hydroxide to give 28 in 51% yield (in 2
steps). Compound 28 was converted to acyl chloride
and coupled with 29 to produce amide 30 in 74%
yield. Compound 30 was refluxed with p-toluenesulf-
onic acid in p-xylene to give 31 by cyclization-dehy-
dration in 32% yield and a demethylation product 32
in 9% yield. Compound 31 was converted to 33 by
saponification. Then, 33 was treated with oxalyl
chloride and N,N-dimethylformamide to produce
the corresponding acyl chloride, which was coupled
with 29 to give the amide 34 in 61% yield (in two
steps). Compound 34 was refluxed with p-toluenesulf-
onic acid in p-xylene to give 35 by cyclization-
dehydration.

6110
S.-T. Huang et al. / Bioorg. Med. Chem. 14 (2006) 6106–6119
Lawesson's
reagent
OBn S
CO2H
OBn
OBn O
1. (COCl)2
DMF, CH2Cl2
N
H
N
H
toluene
CO2CH3
2. 2, pyridine
CH2Cl2
CO2CH3
37
36
10
1. K3Fe(CN)6,
30% NaOH, EtOH
2. CH2N2, ether
OBn
S
CO2CH3
1. (COCl)2
CO2H
DMF, CH2Cl2
N
S
N
1N NaOH
THF
S
N
2. 2, pyridine
CH2Cl2
HN
O
OBn
OBn
H3CO2C
38
40
41
.
BF3 OEt2
EtSH, CH2Cl2
OH
S
N
CO2CH3
39
Scheme 6. Synthesis of 38–41.
a
UK-1 analogues with benzothiazole moiety were pre-
pared according to Scheme 6. Compound 10 was treated
with oxalyl chloride and N,N-dimethylformamide to
produce the corresponding acyl chloride, which was
coupled with 2 to give the amide 36 in 81% yield. Then,
36 was converted to thioamide 37 in 51% yield by treat-
ment with Lawesson’s reagent.14 Thioamide 37 was cyc-
lized to benzothiazoles 38 by Jacobson synthesis with
alkaline potassium ferricyanide. The crude products
contained compounds 38 and 40. Therefore, the crude
products were treated with diazomethane and purified
by flash column chromatography to give 38 in 21%
yield. Compound 39 was prepared by the debenzylation
of 38 using ethanethiol and boron trifluoride-diethyl
etherate. Compound 38 was converted to acid 40 by
saponification. Then, 40 was treated with oxalyl chloride
and N,N-dimethylformamide and the corresponding
acyl chloride was produced;15 it was coupled with 2 to
give the amide 41 in 51% yield (in two steps). Then, an
attempt was made to convert 41 to thioamide by treating
it with Lawesson’s reagent and Jacobson synthesis with
alkaline potassium ferricyanide was used to prepare the
corresponding bis(benzothiazole), but the attempt was
unsuccessful.
Table 1. IC50 (lM) values of tumor cell lines after 72 h continuous
exposure to test compounds
Compound
Tumor type/cell line
A-549b BFTC-905c RDd MES-SAe HeLaf
12
13
14
15
16
17
18
19
23
24
25
26
31
32
33
34
39
41
UK-1
70.0
87.0
74.1
38.2
2.8
53.8
66.4
37.9
9.6
>100 50.6
>100 91.0
67.6
67.3
31.5
52.5
7.1
35.9
17.6
13.8
44.9
26.5
28.0
nd
33.2
52.7
<5
11.9
36.8
29.9
21.4
89.2
98.3
49.6
84.9
>100
54.7
>100
nd
6.6
15.3
9.2
nd
22.2
46.9
>100
>100
>100
50.8
>100
>100
>100
ndg
14.5
29.3
82.1
90.3
75.8
51.4
52.6
67.6
>100
nd
33.2
91.3
>100 63.9
58.5
87.8
>100
41.0
>100 >100
>100 89.4
>100 >100
nd
nd
>100
nd
5.1
87.3
nd
39.1
nd
76.7
nd
81.6
nd
nd
nd
nd
10.7
a IC50 is the concentration that induces 50% growth inhibition com-
pared with untreated control cells.
b human lung carcinoma cell line.
c human bladder carcinoma transitional cell line.
d human rhabdomyosarcoma cell line.
e human uterine sarcoma cell line.
3. Biological assay
The cytotoxicity of UK-1 against P388, B16, and HeLa
cell lines has been reported elsewhere.1 A range of hu-
man cancer cell lines A-549 and HeLa cells were investi-
gated to determine the spectrum of cell lines whose
growth is inhibited by UK-1. The results, presented in
Table 1, indicate that UK-1 exhibits potent anticancer
activity against human lung and epithelial carcinoma
cell lines. The cytotoxicities of the UK-1 analogues to
f Human epithelial carcinoma cell line.
g nd, not determined.
cancer cells were examined in order to elucidate the
structural basis for selective cytotoxicity of the natural
product toward a unique cancer cell. Table 1 summariz-
es the results of cytotoxicity testing against human

S.-T. Huang et al. / Bioorg. Med. Chem. 14 (2006) 6106–6119
6111
A-549, BFTC-905, RD, MES-SA, and HeLa carcinoma
cell lines. UK-1 exhibits potent cytotoxic activity against
the A-549 and HeLa cancer cells lines, but compound 16
is more potent than UK-1 against both A-549 cells (IC50
2.8 lM vs 5.1 lM for UK-1) and HeLa cells (IC50
7.1 lM vs 10.7 lM for UK-1). Compound 16 is the pre-
cursor of compound 18, but compound 18 is less potent
than 16 against A-549 cells (IC50 22.2 lM vs 2.8 lM for
16), MES-SA (IC50 9.2 lM vs <5 lM for 16), and HeLa
cells (IC50 14.5 lM vs 7.1 lM for 16). Only one benz-
imidazole ring was formed in compound 16, and this
skeleton relative to 18 may increase the flexibility to
coordinate with Mg2+ ion, and enhanced cytotoxicity
against cancer cell lines. Compound 26 is the precursor
of UK-1, but compound 26 is less potent than UK-1
against A-549 cells (IC50 50.8 lM vs 5.1 lM for UK-1)
and HeLa cells (IC50 151.4 lM vs 10.7 lM for UK-1).
Even though compound 18 has a bis(benzimidazole)
skeleton and UK-1 has the corresponding bis(benzoxaz-
ole) skeleton, compound 18 is less potent than UK-1
against A-549 cells (IC50 22.2 lM vs 5.1 lM for UK-1)
and HeLa cells (IC50 14.5 lM vs 10.7 lM for UK-1).
Kerwin et al. reported the ability of UK-1 to form metal
ion complexes that can bind to DNA5,17 and inhibit
DNA-processing enzymes,5 which findings indicate that
Mg2+ ion binding by UK-1 may lead to biologically rel-
evant complexes with a specific target in cancer cells.
Therefore, the interaction between bis(benzoxazole)
and the Mg2+ ion may be stronger than that of bis(benz-
imidazole). Interestingly, compounds 15, 17, and 18
selectively exhibit potent cytotoxic activity against the
BFTC-905 cells (IC50 9.6 lM), A-549 cells (IC50
6.6 lM), and MES-SA cells (IC50 9.2 lM), respectively.
tional groups, the positions of –NH2 groups that form
anilide differ, and the conformations of their complexes
formed with Mg2+ differ. This occurrence may imply
that compound 26 is less potent than compound 16
and UK-1, even though it is the precursor of UK-1.
3.1. Conclusion
The natural product of bis(benzoxazole), UK-1, has po-
tential for use against cancer cell lines. Structurally sim-
ilar bis(benzimidazole) 17 and 18 were also found to
exhibit potent anticancer activity. Interestingly, com-
pound 16 is the synthetic precursor of compounds 17-
19 and is more potent than UK-1 against human lung
(A-549) and epithelial (HeLa) carcinoma cell lines.
Therefore, further work with UK-1, compounds 15–
18, and their analogues, must be conducted to determine
whether such targeting is involved in the selective cyto-
toxicity of UK-1 and compounds 15–18, and whether
the promising spectrum of the in vitro anticancer activ-
ity of UK-1 and 15–18 reported herein is also reflected
in vivo.
4. Experimental
4.1. Chemistry
4.1.1. General methods. Melting points were recorded
using a Yanagimoto Micro Melting Point Apparatus
Model-S3 capillary melting point apparatus and are
uncorrected. TLC analysis was carried out on silica gel
60 F254 precoated glass sheets and detected under UV
1
light. H and 13C NMR spectra were recorded employ-
Compounds 14, 25, 32, and 39 have the same skeleton
and differ only in their hetero atoms. Interestingly, the
compound that has benzimidazole moiety exhibits
stronger anticancer activity against cell lines than those
that contain benzoxazole or benzothiazole. Compound
18 is less potent than UK-1 against cancer cell lines.
Compound 18 contains bis(bezoimidazole) moiety, and
UK-1 has bis(benzoxazole) moiety, perhaps because
the steric energies18 of chelating complexes of Mg2+ with
compounds 18 (11.71 kcal/mol) exceed that of UK-1
(9.29 kcal/mol). When ester 14 was hydrolyzed to form
the corresponding acid 15, the power of 15 to inhibit
BFTC-905 was increased. The potency of compound
17 against A-549 is stronger than that of compound
18, while the inhibitive potency against BFTC-905,
RD, and MES-SA of compound 18 exceeds that of com-
pound 17. The hydroxyl group of compound 18 was
converted to benzyl ether in 17. Therefore, the binding
sites in compounds 18 and 17 should differ from each
other in the cell lines, because compound 17 is large
sized and hydrophobic. Compound 16 is the most po-
tent against cancer cell lines herein. The flexibility of
16 may increase its ability to chelate with Mg2+ and to
promote the interaction at the binding site. Unfortu-
nately, compound 26 is the precursor of UK-1 that cor-
responds to the compound 16 as the precursor of
compound 18. It does not powerfully inhibit activity
against cancer cell lines, even though it has good flexibil-
ity. Although compounds 16 and 26 have anilide func-
ing a Bruker AVANCE DPE-300 or VARIAN UNI-
YTINOVA-500 spectrometer, and chemical shifts (d)
are in ppm relative to CHCl3 (7.26 ppm or 77 ppm),
CD3OD (d = 3.30 ppm or 49 ppm), and (CD3)2SO
(2.49 ppm or 39.7 ppm) as internal standard. Infrared
spectra were recorded on a JASCO FT/IR 410 spec-
trometer. Mass spectra were recorded on a Micromass
Platform II or Finnigan/Thermo Quest MAT 95XL
spectrometer. In each case proton NMR showed the
presence of indicated solvent(s). All chemicals and
solvents were purchased from Aldrich Chemical Co.,
Fisher Scientific, or Lancaster.
4.1.1.1. 2-Amino-benzoic acid methyl ester (2). To a
solution of 2-aminobenzoic acid (6.00 g, 44.0 mmol) in
diethyl ether (100 mL) was added a solution of diazome-
thane in diethyl ether at 0 ꢁC until ceased to generate
nitrogen. After adding 1 mL of acetic acid, the reaction
solution was concentrated and the resulting residue was
purified by flash column chromatography using silica gel
as the stationary phase and using ethyl acetate/hexane
(1:49–1:19) as the mobile phase producing compound
2
(6.25 g, 41.0 mmol). Yield: 94%. 1H NMR
(300 MHz, CDCl3, d): 7.87–7.83 (d, J = 8.1 Hz, 1H),
7.29–7.23 (dd, J = 8.4, 7.0 Hz, 1H), 6.67–6.61 (m, 2H),
5.71 (s, 2H), 3.86 (s, 3H). 13C NMR (125.7 MHz,
CDCl3, d): 168.4, 149.6, 134.0, 131.1, 116.9, 116.7,
111.1, 51.5. IR (KBr): 3481, 3371, 1692, 1435, 1274,
752 cmꢀ1. MS m/z: 152 (M++1, 5), 151 (M+, 65), 120

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S.-T. Huang et al. / Bioorg. Med. Chem. 14 (2006) 6106–6119
(31), 119 (100), 92 (50). HRMS-EI (m/z): [M]+ calcd for
C8H9NO2, 151.0633; found, 151.0637.
(m/z): [M]+ calcd for C8H8N2O4, 196.0484; found,
196.0489.
4.1.1.2. 2-Acetylamino-3-nitro-benzoic acid methyl
ester (3) and 2-acetylamino-5-nitro-benzoic acid methyl
ester (4). A solution of compound 2 (6.30 g, 41.0 mmol)
in acetic anhydride (47.0 mL) was stirred at room tem-
perature for 1 h, then a solution of acetic anhydride
(9.6 mL), acetic acid (8.4 mL), and concd nitric acid
(13.7 mL) was added slowly at 5–15 ꢁC. After stirring
at 5–15 ꢁC for 2.5 h, the reaction solution was poured
into ice–water (50 mL) and the precipitate was filtered
to give compound 4 (5.89 g, 25.0 mmol). The filtrate
was diluted with water (50 mL) and stood overnight to
give crystalline 3. The mother liquid was extracted with
dichloromethane, after concentration the residue was
purified by flash column chromatography using silica
gel as the stationary phase and using ethyl acetate/hex-
ane (1:3, 1:1) as the mobile phase producing compound
3 (3.68 g, 15.0 mmol, combined with crystalline 3).
4.1.1.6. 2,3-Diamino-benzoic acid methyl ester (6). A
mixture of compound 5 (1.00 g, 5.1 mmol) and Pd/C
(0.10 g, 10% of Pd on charcoal) in methanol (30 mL) un-
der hydrogen atmosphere (60 psi) was hydrogenated for
3 h. The catalyst was removed by filtration, the filtrate
was concentrated, and the resulting residue was purified
by flash column chromatography using silica gel as the
stationary phase and using ethyl acetate/hexane (3:7)
as the mobile phase producing compound 6 (0.81 g,
4.9 mmol). Yield: 96%. mp: 63–65 ꢁC. 1H NMR
(300 MHz, CDCl3, d): 7.48–7.45 (d, J = 8.1 Hz, 1H),
6.86–6.83 (d, J = 7.5 Hz, 1H), 6.62–6.57 (dd, J = 7.9,
7.7 Hz, 1H), 3.87 (s, 3H). 13C NMR (125.7 MHz,
CDCl3, d): 168.9, 141.1, 134.1, 122.6, 120.6, 116.6,
111.9, 51.5. IR (KBr): 3367, 1693, 1433, 1287, 1234,
733 cmꢀ1. MS m/z: 167 (M++1, 8), 166 (M+, 100), 135
(19), 134 (74), 106 (97), 105 (28). HRMS-EI (m/z):
[M]+ calcd for C8H10N2O2, 166.0742; found, 166.0748.
4.1.1.3. Compound 3. Yield: 37%. mp: 120–122 ꢁC. 1H
NMR (300 MHz, CDCl3, d): 10.34 (s, 1H), 8.22–8.19 (d,
J = 6.3 Hz, 1H), 8.11–8.07 (d, J = 8.2 Hz, 1H), 7.36–7.31
(dd, J = 8.1, 6.5 Hz, 1H), 3.96 (s, 3H), 2.23 (s, 3H). 13C
NMR (125.7 MHz, CDCl3, d): 168.6, 166.6, 144.2,
134.8, 132.1, 129.4, 123.8, 122.6, 53.0, 24.1. IR (KBr):
3307, 1683, 1527, 1438, 1365, 1267, 1120 cmꢀ1. MS
m/z: 238 (M+, 0.58), 196 (100), 192 (15), 165 (12), 164
(53), 90 (14). HRMS-EI (m/z): [M]+ calcd for
C10H10N2O5, 238.0590; found, 238.0591.
4.1.1.7. 2-Hydroxy-benzoic acid methyl ester (8). To a
solution of compound 7 (7.80 g, 57.0 mmol) in diethyl
ether and methanol was added a solution of diazome-
thane in diethyl ether at 0 ꢁC until ceased to generate
nitrogen. After adding a few drops of acetic acid, the
reaction solution was concentrated to produce product
8
(8.60 g, 57.0 mmol). Yield: 100%. 1H NMR
(300 MHz, CDCl3, d): 10.75 (s, 1H), 7.85–7.82 (d,
J = 8.0 Hz, 1H), 7.46–7.42 (dd, J = 8.7, 8.4 Hz, 1H),
6.99–6.96 (d, J = 8.4 Hz, 1H), 6.90–6.85 (dd, J = 8.2,
8.1 Hz, 1H), 3.95 (s, 3H). 13C NMR (125.7 MHz,
CDCl3, d): 170.5, 161.5, 135.6, 129.8, 119.1, 117.5,
112.3, 52.2. IR (KBr): 3186, 1678, 1305, 1090, 845,
757 cmꢀ1. MS m/z: 152 (M+, 49), 121 (27), 120 (100),
92 (53), 65 (16). HRMS-EI (m/z): [M]+ calcd for
C8H8O3, 152.0473; found, 152.0479.
4.1.1.4. Compound 4. Yield: 61%. mp: 177–181 ꢁC. 1H
NMR (300 MHz, CDCl3, d): 11.38 (s, 1H), 8.95–8.92
(m, 2H), 8.39–8.35 (d, J = 9.4 Hz, 1H), 4.0 (s, 3H),
2.29 (s, 3H). 13C NMR (125.7 MHz, CDCl3, d): 169.4,
167.2, 146.5, 141.6, 129.4, 126.8, 120.3, 114.4, 53.0,
25.5. IR (KBr): 3004, 1685, 1587, 1508, 1436, 1348,
902, 821 cmꢀ1. MS m/z: 238 (M+, 18), 196 (100), 164
(45), 119 (12), 57 (28), 55 (15). HRMS-EI (m/z): [M]+
calcd for C10H10N2O5, 238.0590; found, 238.0587.
4.1.1.8. 2-Benzyloxy-benzoic acid methyl ester (9). To
a solution of compound 8 (10.00 g, 28.0 mmol) in anhy-
drous dichloromethane (99.0 mL) and anhydrous meth-
anol (99.0 mL) were added potassium carbonate
(20.00 g, 145.0 mmol) and benzyl bromide (15.70 mL,
132.0 mmol), and refluxed for 40 h under nitrogen atmo-
sphere. After filtration, the filtrate was concentrated,
and the residue was purified by flash column chromatog-
raphy using silica gel as the stationary phase and using
ethyl acetate/hexane (1:49) as the mobile phase produc-
4.1.1.5. 2-Amino-3-nitro-benzoic acid methyl ester (5).
To a solution of compound 3 (0.50 g, 2.1 mmol) in anhy-
drous methanol (25 mL) was added a sodium methoxide
solution (37.0 mL, 0.07 mmol, 1.92 M in methanol) at
room temperature. After the reaction was completed,
added acetic acid to neutralize the reaction solution.
The solvent was removed, and the residue was extracted
by ethyl acetate. The extract was dried by anhydrous
magnesium sulfate and concentrated. The resulting resi-
due was dissolved in a mixture of diethyl ether and
methanol, and added diazomethane at 0 ꢁC until ceased
to generate nitrogen. After adding a few drops of acetic
acid, the reaction solution was concentrated to produce
product 5 (0.40 g, 2.0 mmol). Yield: 87%. mp: 97–
1
ing compound 9 (14.79 g, 61.0 mmol). Yield: 93%. H
NMR (300 MHz, CDCl3, d): 7.84–7.80 (d, J = 7.6 Hz,
1H), 7.51–7.31 (m, 6H), 7.02–6.99 (m, 2H), 5.19 (s,
2H), 3.91 (s, 3H). 13C NMR (125.7 MHz, CDCl3, d):
166.7, 158.0, 136.6, 133.3, 131.3, 128.4, 127.6, 126.6,
120.6, 120.4, 113.7, 70.4, 51.8. IR (KBr): 2949, 1727,
1599, 1450, 1249, 754 cmꢀ1. MS m/z: 242 (M+, 3), 210
(4), 92 (9), 91 (100), 65 (8). HRMS-EI (m/z): [M]+ calcd
for C15H14O3, 242.0943; found, 242.0951.
1
100 ꢁC. H NMR (300 MHz, CD3Cl3, d): 8.40–8.37 (d,
J = 8.4 Hz, 1H), 8.25–8.22 (d, J = 7.7 Hz, 1H), 6.68–
6.63 (dd, J = 8.3, 7.8 Hz, 1H), 3.92 (s, 3H). 13C NMR
(125.7 MHz, CDCl3, d): 167.4, 147.2, 139.3, 133.1,
132.2, 114.3, 113.9, 52.2. IR (KBr): 3120, 3028, 953,
882, 847, 706 cmꢀ1. MS m/z: 197 (M++1, 9), 196 (M+,
100), 165 (46), 119 (24), 118 (16), 90 (41). HRMS-EI
4.1.1.9. 2-Benzyloxy-benzoic acid (10). To a solution
of compound 9 (5.20 g, 21.0 mmol) in 1,4-dioxane
(50 mL) was added 1.4 M sodium hydroxide solution
(22 mL) and refluxed for 2 h. After concentration, the

S.-T. Huang et al. / Bioorg. Med. Chem. 14 (2006) 6106–6119
6113
remained concentrated solution was acidified to pH 1 by
adding 1 N hydrochloric acid. The acidic solution was
extracted with ethyl acetate, and the extract was dried
over anhydrous magnesium sulfate, filtered, and concen-
trated to produce compound 10 (5.00 g, 22.0 mmol).
Yield: 100%. mp: 73–75 ꢁC. 1H NMR (300 MHz,
CDCl3, d): 10.8 (s, 1H), 8.23–8.2 (d, J = 7.8 Hz, 1H),
7.57–7.56 (m, 1H), 7.45–7.42 (m, 5H), 7.18–7.12 (m,
2H), 5.26 (s, 2H). 13C NMR (125.7 MHz, CDCl3, d):
165.3, 157.3, 135.0, 134.2, 133.8, 129.16, 129.12, 127.8,
122.4, 117.9, 113.0, 72.1. IR (KBr): 3048, 2952, 1727,
1600, 1450, 1251, 1083, 755 cmꢀ1. MS m/z: 228 (M+,
1), 92 (11), 91 (100), 69 (9), 57 (15). HRMS-EI (m/z):
[M]+ calcd for C14H12O3, 228.0786; found, 228.0795.
4.1.1.12. 2-(2-Benzyloxy-phenyl)-1H-benzoimidazole-
4-carboxylic acid (13). To a solution of compound 12
(0.37 g, 1.0 mmol) in tetrahydrofuran (7.3 mL) was add-
ed 5 M sodium hydroxide solution (4.5 mL), and re-
fluxed for 12 h. After cooled to room temperature, the
reaction solution was acidified by concd hydrochloric
acid to give a white precipitate. The precipitate was fil-
tered and washed with water, and dried under vacuum
to produce compound 13 (0.34 g, 1.0 mmol). Yield:
1
97%. mp: 180–182 ꢁC. H NMR (300 MHz, CD3OD,
d): 8.22–8.19 (d, J = 7.6 Hz, 1H), 8.07–8.05 (d,
J = 8 Hz, 2H), 7.79–7.07 (m, 2H), 7.51–7.48 (m, 3H),
7.36–7.29 (m, 4H), 5.46 (s, 2H). 13C NMR
(125.7 MHz, CDCl3, d): 167.1, 158.9, 150.0, 137.0,
136.8, 133.5, 131.8, 131.5, 129.8, 129.5, 129.2, 128.9,
127.3, 123.1, 119.8, 118.4, 115.7, 112.5, 72.8. IR (KBr):
3351, 2607, 1697, 1128, 848, 622 cmꢀ1. MS m/z: 345
(M++1, 8), 344 (M+, 39), 298 (8), 238 (8), 236 (9), 91
(100). HRMS-EI (m/z): [M]+ calcd for C21H16N2O3,
344.1161; found, 344.1159.
4.1.1.10. 2-Amino-3-(2-benzyloxy-benzoylamino)-ben-
zoic acid methyl ester (11). To a solution of compound
10 (1.40 g, 6.0 mmol) and compound
6 (0.50 g,
3.0 mmol) in anhydrous dichloromethane (15 mL) were
added dicyclohexylcarbodiimide (1.23 g, 6.0 mmol) and
4-(dimethylamino)pyridine (12 mg) under argon atmo-
sphere, and stirred at room temperature for 7 h. The
reaction mixture was filtered through celite, and the fil-
trate was concentrated. The residue was purified by flash
column chromatography using silica gel as the station-
ary phase and using ethyl acetate/hexane (1:19, 1:9,
3:17) as the mobile phase producing compound 11
(0.94 g, 25.0 mmol). Yield: 83%. mp: 115–117 ꢁC. 1H
NMR (300 MHz, CDCl3, d): 9.16 (s, 1H), 8.28–8.26
(d, J = 7.8 Hz, 1H), 7.76–7.73 (d, J = 8.1 Hz, 1H),
7.54–7.44 (m, 7H), 7.18–7.13 (m, 2H), 6.67–6.62 (dd,
J = 7.9, 7.9 Hz, 1H), 5.56 (s, 2H), 5.26 (s, 2H), 3.85 (s,
3H). 13C NMR (75.4 MHz, CDCl3, d): 168.4, 164.0,
156.6, 145.0, 135.1, 133.3, 132.6, 130.1, 129.1, 128.8,
128.6, 124.6, 121.9, 121.8, 115.8, 112.7, 112.3, 71.7,
51.6. IR (KBr): 3282, 2935, 1631, 1446, 1243, 1091,
746 cmꢀ1. MS m/z: 377 (M++1, 5), 376 (M+, 24), 211
(20), 165 (16), 121 (12). HRMS-EI (m/z): [M]+ calcd
for C22H20N2O4, 376.1423; found, 376.1419.
4.1.1.13. 2-(2-Hydroxy-phenyl)-1H-benzoimidazole-4-
carboxylic acid methyl ester (14). A mixture of com-
pound 12 (0.21 g, 0.6 mmol) and Pd/C (0.02 g, 10% of
Pd on charcoal) in ethyl acetate (10 mL) under hydrogen
atmosphere (60 psi) was hydrogenated for 2 h. The cat-
alyst was removed by filtration, and the filtrate was con-
centrated to give compound 14 (0.15 g, 0.6 mmol).
Yield: 97%. mp: 205–207 ꢁC. 1H NMR (300 MHz,
CD3Cl3, d): 10.72 (s, 1H), 7.96–7.91 (dd, J = 8.2,
7.8 Hz, 2H), 7.70–7.67 (d, J = 7.8 Hz, 1H), 7.43–7.32
(m, 2H), 7.15–7.12 (d, J = 8.1 Hz, 1H), 7.02–6.97 (dd,
J = 7.8, 7.3 Hz, 1H), 4.04 (s, 3H). 13C NMR
(75.4 MHz, CDCl3, d): 167, 159.1, 152.2, 142.4, 133.4,
125.0, 123.9, 122.3, 119.2, 118.1, 113.2, 111.7, 52.3. IR
(KBr): 3394, 1702, 1463, 1284, 750 cmꢀ1. MS m/z: 269
(M++1, 8), 268 (M+, 49), 237 (17), 236 (100), 208 (18).
HRMS-EI (m/z): [M]+ calcd for C15H12N2O3,
268.0848; found, 268.0843.
4.1.1.11. 2-(2-Benzyloxy-phenyl)-1H-benzoimidazole-
4-carboxylic acid methyl ester (12). A solution of com-
pound 11 (0.48 g, 1.3 mmol) in acetic acid (6 mL) was
refluxed at 120–130 ꢁC for 3 h. After cooled to room
temperature, the reaction solution was poured into ice
water and neutralized by sodium bicarbonate. The aque-
ous solution was extracted with dichloromethane, and
the extract was washed with brine and dried over anhy-
drous magnesium sulfate. The dry solution was concen-
trated and dried under vacuum to give compound 12
(0.40 g, 1.1 mmol). Yield: 87%. mp: 103–105 ꢁC. 1H
NMR (300 MHz, CDCl3, d): 11.45 (s, 1H), 8.62–8.59
(d, J = 6.4 Hz, 1H), 8.03–8 (d, J = 7.7 Hz, 1H), 7.9–
7.87 (d, J = 1 Hz, 1H), 7.56–7.53 (m, 2H), 7.46–7.42
(m, 4H), 7.34–7.29 (m, 1H), 7.21–7.16 (m, 2H), 5.36 (s,
2H), 3.66 (s, 3H). 13C NMR (75.4 MHz, CDCl3, d):
166.3, 156.4, 150.9, 143.8, 135.7, 134.2, 131.6, 130.5,
128.9, 128.5, 128.0, 124.5, 124.3, 121.9, 121.7, 118.0,
113.4, 113.0, 71.3, 45.1. IR (KBr): 3437, 1719, 1598,
1461, 1286, 994, 746 cmꢀ1. MS m/z: 359 (M++1, 7),
358 (M+, 33), 252 (14), 207 (11), 91 (100). HRMS-EI
(m/z): [M]+ calcd for C22H18N2O3, 358.1317; found,
358.1324.
4.1.1.14. 2-(2-Hydroxy-phenyl)-1H-benzoimidazole-4-
carboxylic acid (15). A mixture of compound 13 (0.20 g,
0.6 mmol) and Pd/C (0.02 g, 10% of Pd on charcoal) in
methanol (15 mL) under hydrogen atmosphere (60 psi)
was hydrogenated for 2 h. The catalyst was removed
by filtration, and the filtrate was concentrated, and the
resulting residue was purified by flash column chroma-
tography using silica gel as the stationary phase and
using methanol/ethyl acetate (1:4) as the mobile phase
producing compound 15 (0.09 g, 0.4 mmol). Yield:
1
60%. mp: 310 ꢁC (decomposed). H NMR (300 MHz,
DMSO-d6, d): 8.26 (b, 1H), 7.76–7.74 (b, 2H), 7.34–
7.28 (b, 1H), 7.24–7.16 (b, 2H), 6.91 (b, 1H). 13C
NMR (125.7 MHz, DMSO, d): 169.1, 157.6, 151.5,
141.2, 134.0, 131.6, 127.7, 124.2, 121.5, 120.3, 119.0,
117.1, 113.5. IR (KBr): 3396, 1585, 1488, 1384, 1292,
1257, 748 cmꢀ1. MS m/z: 255 (M++1, 6), 254 (M+, 43),
237 (15), 236 (100), 208 (20). HRMS-EI (m/z): [M]+
calcd for C14H10N2O3, 254.0691; found, 254.0684.
4.1.1.15. 2-Amino-3-{[2-(2-benzyloxy-phenyl)-1H-ben-
zoimidazole-4-carbonyl]-amino}-benzoic acid methyl ester
(16). A solution of compound 13 (0.34 g, 1.0 mmol) in

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S.-T. Huang et al. / Bioorg. Med. Chem. 14 (2006) 6106–6119
thionyl chloride (2 mL) was refluxed for 1 h, and the
thionyl chloride was removed by distillation. The re-
mained residue was dissolved in anhydrous dichloro-
methane, to this solution was added a solution of
compound 6 (0.17 g, 1.0 mmol), triethylamine (1.0 mL)
in anhydrous dichloromethane (5 mL) by canula. The
resulting solution was stirred at room temperature over-
night and quenched by adding water. The aqueous solu-
tion was extracted with dichloromethane, and the
extract was dried over anhydrous magnesium sulfate.
The organic solution was concentrated, and the residue
was purified by flash column chromatography using sil-
ica gel as the stationary phase and using methanol/ethyl
acetate (1:1, 3:1) as the mobile phase producing com-
pound 16 (0.27 g, 0.6 mmol). Yield: 56%. mp: 178–
181 ꢁC. 1H NMR (300 MHz, CD3OD, d): 11.68 (b,
1H), 10.84 (b, 1H), 8.63–8.60 (d, J = 9.0 Hz, 1H),
8.21–8.18 (d, J = 7.3 Hz, 1H), 8.07–8.05 (d, J = 7.4 Hz,
1H), 7.82–7.79 (d, J = 8.0 Hz, 1H), 7.52–7.14 (m,
10H), 6.81–6.76 (dd, J = 7.4, 7.3 Hz, 1H), 6.32 (b, 2H),
5.32 (s, 2H), 3.91 (s, 3H). 13C NMR (75.4 MHz, CDCl3,
d): 168.8, 164.3, 156.4, 150.2, 144.4, 140.4, 135.7, 133.6,
132.1, 130.4, 129.24, 129.21, 129.0, 127.9, 127.8, 125.6,
124.3, 122.7, 122.4, 122.3, 117.0, 116.0, 114.6, 113.0,
111.9, 71.5, 51.6. IR (KBr): 3392, 1704, 1673, 1292,
1249, 742 cmꢀ1. MS m/z: 493 (M++1, 18), 492 (M+,
54), 475 (44), 474 (42), 327 (57). HRMS-EI (m/z): [M]+
calcd for C29H24N4O4, 492.1798; found, 492.1792.
8.12–8.09 (d, J = 8.1 Hz, 1H), 7.97–7.95 (d, J = 7.9 Hz,
1H), 7.89–7.86 (m, 2H), 7.77–7.74 (d, J = 7.8 Hz, 1H),
7.46–7.37 (m, 3H), 7.17–7.15 (d, J = 7.5 Hz, 1H), 7.09–
7.04 (dd, J = 7.8, 7.4 Hz, 1H), 4.06 (s, 3H). 13C NMR
(125.7 MHz, DMSO-d6, d): 165.9, 156.2, 152.0, 151.6,
144.3, 140.1, 135.8, 133.8, 131.9, 129.1, 124.2, 124.1,
122.4, 121.7, 120.1, 119.6, 116.8, 115.0, 113.3, 52.3. IR
(KBr): 3486, 3378, 3189, 3068, 2977, 2817, 1675, 1608,
1583, 784 cmꢀ1. MS m/z: 385 (M++1, 25), 384 (100),
353 (24), 352 (93), 162 (13). HRMS-EI (m/z): [M]+ calcd
for C22H16N4O3, 384.1222; found, 384.1219.
4.1.1.18. 20-(2-Benzyloxy-phenyl)-1H,10H-[2,40]biben-
zoimidazolyl-4-carboxylic acid (19). To a solution of
compound 17 (0.05 g, 0.1 mmol) in tetrahydrofuran
(2 mL) was added 5 M sodium hydroxide solution
(3 mL), and refluxed for 48 h. After cooled to room tem-
perature, the reaction solution was acidified by concen-
trated hydrochloric acid to give a white precipitate. The
precipitate was filtered and washed with water, and
dried under vacuum to produce compound 19 (0.08 g,
1
0.2 mmol). Yield: 100%. mp: 256 ꢁC (decomposed). H
NMR (300 MHz, CD3OD, d): 8.55–8.53 (d,
J = 7.5 Hz, 1H), 8.02–7.99 (d, J = 7.5 Hz, 2H), 7.88–
7.85 (d, J = 9.0 Hz, 1H), 7.78–7.76 (d, J = 6.0 Hz, 6H),
7.56–7.28 (m, 8H), 7.16–7.13 (d, J = 8.4 Hz, 1H), 7.06–
7.02 (dd, J = 7.5, 6.0 Hz, 1H), 5.33 (s, 2H). 13C NMR
(125.7 MHz, d-DMSO, d): 166.6, 155.9, 151.1, 149.8,
140.2, 136.9, 135.4, 132.4, 131.7, 130.3, 128.5, 127.7,
127.1, 126.2, 124.3, 123.0, 121.6, 120.7, 116.9, 115.8,
114.1, 69.1. IR (KBr): 3390, 3200, 3095, 3050, 1625,
1575, 1550, 1095, 750 cmꢀ1. MS m/z: 460 (M+, 1.78),
418 (45), 253 (14), 252 (100), 91 (26). HRMS-EI (m/z):
[M]+ calcd for C28H20N4O3, 460.1535; found, 460.1541.
4.1.1.16. 20-(2-Benzyloxy-phenyl)-1H,10H-[2,40]biben-
zoimidazolyl-4-carboxylic acid methyl ester (17). A solu-
tion of compound 16 (0.17 g, 0.4 mmol) in acetic acid
(3 mL) was refluxed at 120–130 ꢁC for 2 h. After cooled
to room temperature, the reaction solution was poured
into ice water and neutralized by sodium bicarbonate.
The aqueous solution was extracted with dichlorometh-
ane, and the extract was washed with brine and dried
over anhydrous magnesium sulfate. The dry solution
was concentrated and dried under vacuum to give com-
pound 17 (0.14 g, 0.3 mmol). Yield: 85%. mp: 202–
4.1.1.19. 2-Amino-3-hydroxy-benzoic acid methyl ester
(20). To a solution of 2-amino-3-hydroxybenzoic acid
(0.15 g, 0.9 mmol) in diethyl ether (10.0 mL) and meth-
anol (2.0 mL) was added a solution of diazomethane
in diethyl ether at 0 ꢁC until ceased to generate nitrogen.
After adding 1 mL of acetic acid, the reaction solution
was concentrated and the resulting residue was purified
by flash column chromatography using silica gel as the
stationary phase and using ethyl acetate/hexane (1:9)
as the mobile phase producing compound 20 (0.15 g,
0.8 mmol). Yield: 92%. mp: 94–97 ꢁC. 1H NMR
(300 MHz, CDCl3, d): 7.50–7.47 (d, J = 8.7 Hz, 1H),
6.83–6.81 (d, J = 6.6 Hz, 1H), 6.52–6.47 (dd, J = 8.0,
7.9 Hz, 1H), 3.87 (s, 3H). 13C NMR (125.7 MHz,
CDCl3, d): 168.7, 143.1, 140.5, 123.1, 117.9, 115.0,
111.3, 51.5. IR (KBr): 3409, 1704, 1439, 1293,
743 cmꢀ1. MS m/z: 167 (M+, 100), 136 (26), 135 (79),
108 (12), 107 (85). HRMS-EI (m/z): [M]+ calcd for
C8H9NO3, 167.0582; found, 167.0589.
1
206 ꢁC. H NMR (300 MHz, CDCl3, d): 13.66 (s, 1H),
10.74 (s, 1H), 9.01–8.98 (d, J = 7.8 Hz, 1H), 8.40–8.37
(m, 1H), 8.07–8.04 (d, J = 8.0 Hz, 1H), 7.99–7.96 (d,
J = 7.7 Hz, 1H), 7.54–7.49 (m, 6H), 7.37–7.31 (m, 4H),
7.22–7.19 (d, J = 8.8 Hz, 1H), 5.36 (s, 2H), 4.18 (s,
3H). 13C NMR (75.4 MHz, CDCl3, d): 166.6, 156.2,
152.4, 150.2, 144.6, 140.5, 135.8, 134.3, 133.8, 131.7,
130.4, 129.1, 128.9, 127.8, 124.7, 124.2, 122.9, 121.8,
121.4, 120.8, 118.5, 117.3, 113.6, 112.6, 112.5, 71.2,
52.0. IR (KBr): 3413, 1720, 1432, 1280, 744 cmꢀ1. MS
m/z: 475 (M++1, 33), 474 (M+, 100), 473 (20), 352
(25), 351 (33). HRMS-EI (m/z): [M]+ calcd for
C29H22N4O3, 474.1692; found, 474.1684.
4.1.1.17. 20-(2-Hydroxy-phenyl)-1H,10H-[2,40]bibenzo-
imidazolyl-4-carboxylic acid methyl ester (18). A mixture
of compound 17 (0.05 g, 0.1 mmol) and Pd/C (2.5 mg,
10% of Pd on charcoal) in methanol (25 mL) under
hydrogen atmosphere (1 atm) was hydrogenated over-
night. The catalyst was removed by filtration, and the fil-
trate was concentrated to give compound 18 (0.03 g,
0.07 mmol). Yield: 66%. mp: 283–286 ꢁC. 1H NMR
(300 MHz, CDCl3, d): 12.0 (s, 1H), 10.88 (s, 1H),
4.1.1.20.
2-(2-Benzyloxy-benzoylamino)-3-hydroxy-
benzoic acid methyl ester (22). A solution of compound
10 (0.28 g, 1.2 mmol) in anhydrous dichloromethane
(10 mL) was added oxalyl chloride (0.22 mL, 2.5 mmol),
and stirred for 10 min at room temperature; following
added 5 drops of N,N-dimethylformamide and stirred
for 2 h. Then the solvent was removed and dried under
vacuum for 3 h. The residue was dissolved in anhydrous

S.-T. Huang et al. / Bioorg. Med. Chem. 14 (2006) 6106–6119
6115
dichloromethane (10 mL). To this solution was added a
solution of compound 20 (0.15 g, 0.8 mmol) and pyri-
dine (2 mL) in anhydrous dichloromethane (10 mL) via
canula, and stirred overnight. The reaction was
quenched by adding water and extracted with dichloro-
methane. The organic phase was dried over anhydrous
magnesium sulfate. After filtration and concentration,
the resulting residue was purified by flash column chro-
matography using silica gel as the stationary phase and
using ethyl acetate/hexane (1:19, 1:9) as the mobile
phase producing compound 22 (0.30 g, 0.8 mmol).
Yield: 96%. mp: 101–102 ꢁC. 1H NMR (300 MHz,
CDCl3, d): 12.03 (s, 1H), 9.39 (s, 1H), 8.26–8.23 (d,
J = 7.9 Hz, 1H), 7.60–7.57 (d, J = 7.7 Hz, 1H), 7.47–
7.40 (m, 3H), 7.36-7.28 (m, 4H), 7.23–7.18 (m, 1H),
7.11–7.05 (dd, J = 7.9, 7.9 Hz, 1H), 7.04–7.01 (d,
J = 8.3 Hz, 1H), 5.47 (s, 2H), 3.78 (s, 3H). 13C NMR
(125.7 MHz, CDCl3, d): 167.5, 165.5, 157.0, 150.7,
136.2, 133.9, 132.7, 128.6, 128.2, 128.0, 126.9, 126.0,
125.7, 122.9, 121.5, 121.3, 120.9, 113.3, 70.8, 52.2. IR
(KBr): 1710, 1627, 1455, 1288, 989, 751 cmꢀ1. MS m/z:
377 (M+, 22), 211 (74), 166 (22), 121 (17), 91 (100).
HRMS-EI (m/z): [M]+ calcd for C22H19NO5, 377.1263;
found, 377.1257.
m/z: 346 (M++1, 13), 345 (M+, 59), 328 (9), 237 (10),
91 (100). HRMS-EI (m/z): [M]+ calcd for C21H15NO4,
345.1001; found, 345.0995.
4.1.1.23. 2-(2-Hydroxy-phenyl)-benzoxazole-4-carbox-
ylic acid methyl ester (25). A solution of compound 23
(0.05 g, 0.1 mmol) and p-toluenesulfonic acid (0.06 g,
0.3 mmol) in anhydrous toluene (2 mL) was refluxed
for 1.5 h, and the reaction was quenched by adding sat-
urated sodium bicarbonate solution. The aqueous phase
was extracted with ethyl acetate, and the extract was
dried over anhydrous magnesium sulfate. After filtration
and concentration, the residue was purified by flash col-
umn chromatography using silica gel as the stationary
phase and using ethyl acetate/hexane (1:49, 1:19) as
the mobile phase producing compound 25 (0.02 g,
0.07 mmol). Yield: 51%. mp: 131–135 ꢁC. 1H NMR
(300 MHz, CDCl3, d): 11.9 (s, 1H), 8.09–8.02 (m, 2H),
7.82–7.79 (d, J = 8.0 Hz, 1H), 7.51–7.43 (m, 2H), 7.16–
7.13 (d, J = 7.9 Hz, 1H), 7.05–7.0 (dd, J = 7.9, 7.6 Hz,
1H), 4.06 (s, 3H). 13C NMR (125.7 MHz, CDCl3, d):
165.5, 164.3, 159.3, 149.7, 139.4, 134.2, 127.4, 127.2,
124.7, 121.2, 119.2, 117.6, 114.9, 109.9, 52.4. IR (KBr):
1717, 1633, 1548, 1301, 747 cmꢀ1. MS m/z: 269 (M+,
62), 238 (23), 237 (100), 211 (36), 209 (15), 133 (13).
HRMS-EI (m/z): [M]+ calcd for C15H11NO4, 269.0688;
found, 269.0684.
4.1.1.21. 2-(2-Benzyloxy-phenyl)-benzoxazole-4-car-
boxylic acid methyl ester (23). Compound 22 (0.11 g,
0.3 mmol) was heated up to 230 ꢁC under vacuum (ca.
40 torr) for 5 h, and purified by flash column chroma-
tography using silica gel as the stationary phase and
using ethyl acetate/hexane (1:49, 1:19) as the mobile
phase producing compound 23 (0.09 g, 0.3 mmol).
Yield: 86%. mp: 104–105 ꢁC. 1H NMR (300 MHz,
CDCl3, d): 8.28–8.26 (d, J = 7.9 Hz, 1H), 8.06–8.03 (d,
J = 7.8 Hz, 1H), 7.77–7.75 (d, J = 7.6 Hz, 1H), 7.67–
7.64 (d, J = 7.2 Hz, 2H), 7.54–7.25 (m, 5H), 7.15–7.10
(m, 2H), 5.31 (s, 2H), 4.01 (s, 3H). 13C NMR
(125.7 MHz, CDCl3, d): 165.9, 163.6, 157.7, 151.2,
141.3, 136.6, 133.2, 131.9, 128.4, 127.7, 126.87, 126.83,
124.1, 121.9, 121.0, 116.2, 114.7, 113.6, 70.6, 52.4. IR
(KBr): 1703, 1601, 1450, 1240, 752 cmꢀ1. MS m/z: 360
(M++1, 19), 359 (M+, 80), 342 (15), 327 (16), 91 (100).
HRMS-EI (m/z): [M]+ calcd for C22H17NO4, 359.1158;
found, 359.1165.
4.1.1.24. 2-{[2-(2-Benzyloxy-phenyl)-benzoxazole-4-
carbonyl]-amino}-3-hydroxy-benzoic acid methyl ester
(26). A solution of compound 24 (0.07 g, 0.2 mmol)
in anhydrous dichloromethane (10 mL) was added
oxalyl chloride (0.15 mL, 1.7 mmol), and stirred for
10 minutes at room temperature; following added 5
drops of N,N-dimethylformamide, and stirred for
2 h. Then the solvent was removed and dried under
vacuum for 3 h. The residue was dissolved in anhy-
drous dichloromethane (15 mL). To this solution was
added a solution of compound 20 (0.04 g, 0.2 mmol)
and pyridine (0.5 mL) in anhydrous dichloromethane
(6 mL) via canula, and stirred overnight. The reaction
was quenched by adding water and extracted with
dichloromethane. The organic phase was dried over
anhydrous magnesium sulfate. After filtration and
concentration, the resulting residue was purified by
flash column chromatography using silica gel as the
stationary phase and using ethyl acetate/hexane (1:9)
as the mobile phase producing compound 26 (0.08 g,
0.2 mmol). Yield: 76%. mp: 155–158 ꢁC. 1H NMR
(300 MHz, CDCl3, d): 12.57 (s, 1H), 8.80 (s, 1H),
8.49–8.46 (d, J = 7.7 Hz, 1H), 8.30–8.27 (d,
J = 7.8 Hz, 1H), 7.80–7.77 (d, J = 8.1 Hz, 1H), 7.64–
7.61 (d, J = 7.7 Hz, 1H), 7.53–7.50 (m, 4H), 7.37–
7.27 (m, 4H), 7.23–7.21 (m, 1H), 7.19–7.10 (m, 1H),
5.24 (s, 2H), 3.77 (s, 3H). 13C NMR (125.7 MHz,
CDCl3, d): 167.1, 164.4, 163.4, 157.9, 151.2, 150.8,
139.9, 136.2, 133.6, 132.3, 128.3, 127.8, 127.6, 126.7,
126.5, 126.3, 125.6, 124.7, 123.6, 123.0, 122.5, 121.2,
115.7, 114.7, 113.7, 70.7, 52.2. IR (KBr): 3208, 1713,
1527, 1458, 1291, 750 cmꢀ1. MS m/z: 494 (M+, 39),
328 (100), 238 (48), 237 (31), 91 (25). HRMS-EI
(m/z): [M]+ calcd for C29H22N2O6, 494.1478; found,
494.1482.
4.1.1.22. 2-(2-Benzyloxy-phenyl)-benzoxazole-4-car-
boxylic acid (24). To a solution of compound 23
(0.09 g, 0.3 mmol) in tetrahydrofuran (2 mL) was added
5 N sodium hydroxide solution (1 mL), and refluxed for
2 h. After cooled to room temperature, the reaction
solution was acidified by concd hydrochloric acid and
extracted with ethyl acetate. The extract was dried over
anhydrous magnesium sulfate. After filtration and con-
centration, the residue was dried under vacuum to give
product 24 (0.07 g, 0.2 mmol). Yield: 84%. mp: 99–
1
102 ꢁC. H NMR (300 MHz, CDCl3, d): 11.75 (s, 1H),
8.25–8.22 (d, J = 7.9 Hz, 1H), 8.15–8.12 (d, J = 7.8 Hz,
1H), 7.80–7.77 (d, J = 8.1 Hz, 1H), 7.61–7.34 (m, 7H),
7.21–7.14 (m, 2H), 5.34 (s, 2H). 13C NMR
(125.7 MHz, CDCl3, d): 165.1, 163.0, 158.1, 149.8,
141.0, 136.0, 134.3, 131.6, 128.8, 128.2, 127.2, 126.9,
126.2, 125.3, 121.1, 119.9, 115.2, 114.4, 113.6, 70.8. IR
(KBr): 1745, 1604, 1430, 1251, 1020, 746 cmꢀ1. MS

6116
S.-T. Huang et al. / Bioorg. Med. Chem. 14 (2006) 6106–6119
4.1.1.25. 20-(2-Hydroxy-phenyl)-[2,40]bibenzoxazolyl-
135 (29), 123 (51), 105 (78), 79 (19), 77 (33). HRMS-
EI (m/z): [M]+ calcd for C8H8O3, 152.0473; found,
152.0471.
4-carboxylic acid methyl ester (UK-1). A solution of
compound 26 (0.06 g, 0.1 mmol) and p-toluenesulfonic
acid (0.06 g, 0.3 mmol) in anhydrous p-xylene (2 mL)
was refluxed for 2 h, and the reaction was quenched
by adding saturated sodium bicarbonate solution. The
aqueous phase was extracted with ethyl acetate, and
the extract was dried over anhydrous magnesium sul-
fate. After filtration and concentration, the residue was
purified by flash column chromatography using silica
gel as the stationary phase and using ethyl acetate/hex-
ane (1:19, 1:9) as the mobile phase producing UK-1
(0.02 g, 0.1 mmol). Yield: 43%. mp: 209–210 ꢁC. 1H
NMR (300 MHz, CDCl3, d): 12.06 (s, 1H), 8.40–8.37
(d, J = 7.7 Hz, 1H), 8.13–8.11 (d, J = 7.3 Hz, 1H),
8.10–8.07 (d, J = 7.9 Hz, 1H), 7.90–7.88 (d, J = 8.0 Hz,
1H), 7.83–7.80 (d, J = 8.2 Hz, 1H), 7.58–7.46 (m, 3H),
7.21–7.18 (d, J = 8.3 Hz, 1H), 7.05–7.0 (t, J = 7.7,
7.2 Hz, 1H), 4.13 (s, 3H). 13C NMR (125.7 MHz,
CDCl3, d): 166.1, 164.5, 161.8, 159.5, 151.2, 149.8,
141.4, 138.6, 134.2, 127.4, 127.1, 125.28, 125.25, 124.8,
122.3, 119.5, 117.7, 117.5, 115.0, 113.8, 109.9, 52.6. IR
(KBr): 2923, 1724, 1546, 1427, 1259, 744 cmꢀ1. MS m/
z: 387 (M++1, 24), 386 (M+, 100), 355 (26), 354 (83),
328 (28). HRMS-EI (m/z): [M]+ calcd for C22H14N2O5,
386.0903; found, 386.0904.
4.1.1.28.
2-Hydroxy-3-(2-methoxy-benzoylamino)-
benzoic acid methyl ester (30). A solution of compound
28 (0.15 g, 1.0 mmol) in anhydrous dichloromethane
(15 mL) was added oxalyl chloride (0.26 mL,
3.0 mmol), and stirred for 10 minutes at room temper-
ature; following added 5 drops of N,N-dimethylform-
amide, and stirred for 2 h. Then the solvent was
removed and dried under vacuum for 3 h. The residue
was dissolved in anhydrous dichloromethane (7 mL).
To this solution was added a solution of compound
29 (0.15 g, 0.9 mmol) and pyridine (1 mL) in anhy-
drous dichloromethane (10 mL) via canula, and stirred
overnight. The reaction was quenched by adding
water and extracted with dichloromethane. The organ-
ic phase was dried over anhydrous magnesium sulfate.
After filtration and concentration, the resulting residue
was purified by flash column chromatography using
silica gel as the stationary phase and using ethyl ace-
tate/hexane (3:17, 3:7) as the mobile phase producing
compound 30 (0.20 g, 0.7 mmol). Yield: 74%. mp:
166–168 ꢁC. 1H NMR (300 MHz, CDCl3, d): 11.35
(s, 1H), 10.77 (s, 1H), 8.86–8.83 (d, J = 8.0 Hz, 1H),
8.32–8.29 (d, J = 7.9 Hz, 1H), 7.58–7.54 (d,
J = 8.3 Hz, 1H), 7.53–7.47 (m, 1H), 7.16–7.10 (dd,
J = 7.5, 7.5 Hz, 1H), 7.06–7.03 (d, J = 8.3 Hz, 1H),
6.96–6.91 (dd, J = 8.3, 8.1 Hz, 1H), 4.09 (s, 3H),
3.98 (s, 3H). 13C NMR (125.7 MHz, CDCl3, d):
171.0, 163.2, 157.4, 150.9, 133.2, 132.3, 128.3, 125.5,
123.4, 121.7, 121.4, 119.2, 111.5, 56.1, 52.4. IR
(KBr): 1665, 1536, 1344, 1286, 756 cmꢀ1. MS m/z:
302 (M++1, 5), 301 (M+, 31), 269 (6), 136 (8), 135
(100). HRMS-EI (m/z): [M]+ calcd for C16H15NO5,
301.0950; found, 301.0946.
4.1.1.26. 2-Methoxy-benzoic acid methyl ester (27). To
a solution of salicylic acid 7 (2.20 g, 16.0 mmol) in
anhydrous acetone (30 mL) were added potassium car-
bonate (5.53 g, 40.0 mmol) and iodomethane (2.18 mL,
35.0 mmol), and refluxed overnight. After cooled to
room temperature, the reaction mixture was filtered
through Celite, and the filtrate was concentrated.
The residue was purified by flash column chromatog-
raphy using silica gel as the stationary phase and
using ethyl acetate/hexane (1:19, 1:9) as the mobile
phase producing compound 27 (2.40 g, 14.0 mmol).
Yield: 91%. 1H NMR (300 MHz, CDCl3, d): 7.81–
7.78 (d, J = 7.9 Hz, 1H), 7.50–7.44 (m, 1H), 7.0–6.95
(m, 2H), 3.91 (s, 3H), 3.89 (s, 3H). 13C NMR
(125.7 MHz, CDCl3, d): 166.6, 159.0, 133.5, 131.6,
120.0, 119.9, 111.9, 55.9, 51.9. IR (KBr): 1728, 1492,
1254, 1085, 756 cmꢀ1. MS m/z: 166 (M+, 38), 135
(100), 133 (33), 105 (14), 92 (16), 77 (41). HRMS-EI
(m/z): [M]+ calcd for C9H10O3, 166.0630; found,
166.0632.
4.1.1.29.
2-(2-Methoxy-phenyl)-benzoxazole-7-car-
boxylic acid methyl ester (31) and 2-(2-hydroxy-phenyl)-
benzoxazole-7-carboxylic acid methyl ester (32). A solu-
tion of compound 30 (0.24 g, 0.8 mmol) and p-toluene-
sulfonic acid (0.38 g, 2.0 mmol) in anhydrous p-xylene
(9.7 mL) was refluxed for 4 days, and the reaction was
quenched by adding saturated sodium bicarbonate solu-
tion. The aqueous phase was extracted with ethyl ace-
tate, and the extract was washed with brine and dried
over anhydrous magnesium sulfate. After filtration and
concentration, the residue was dried under vacuum to
produce compound 31 (0.11 g, 0.07 mmol) and com-
pound 32 (20 mg, 0.07 mmol).
4.1.1.27. 2-Methoxy-benzoic acid (28). To a solution
of compound 27 (2.40 g, 14.0 mmol) in methanol
(15 mL) was added 6 M sodium hydroxide solution
(10 mL), and refluxed for 3 h. After concentration, the
remained concentrated solution was acidified by adding
6 N hydrochloric acid to give a precipitate. The precip-
itate was filtered and dried under vacuum to produce
compound 28 (1.71 g, 11.0 mmol). Yield: 56%. mp:
4.1.1.30. Compound 31. Yield: 32%. mp: 88–90 ꢁC. 1H
NMR (300 MHz, CDCl3, d): 8.26–8.23 (d, J = 7.7 Hz,
1H), 8.05–7.99 (m, 2H), 7.57–7.52 (m, 1H), 7.44–7.39
(dd, J = 7.9, 7.9 Hz, 1H), 7.19–7.10 (m, 1H), 4.07 (s,
3H), 4.05 (s, 3H). 13C NMR (125.7 MHz, CDCl3, d):
164.7, 162.3, 158.7, 149.0, 143.3, 133.3, 131.4, 127.0,
124.9, 124.1, 120.8, 115.3, 114.6, 112.1, 56.2, 52.3. IR
(KBr): 1716, 1621, 1428, 1301, 1037, 740 cmꢀ1. MS m/
z: 283 (M+, 70), 252 (21), 251 (37), 224 (17), 223 (100),
167 (16). HRMS-EI (m/z): [M]+ calcd for C16H13NO4,
283.0845; found, 283.0852.
1
108–111 ꢁC. H NMR (300 MHz, CDCl3, d): 10.97 (s,
1H), 8.21–8.17 (d, J = 7.8 Hz, 1H), 7.61–7.55 (dd,
J = 8.6, 7.2 Hz, 1H), 7.17–7.12 (dd, J = 7.4, 7.1 Hz,
1H), 7.08–7.05 (d, J = 8.4 Hz, 1H), 4.08 (s, 3H). 13C
NMR (125.7 MHz, CDCl3, d): 165.3, 158.0, 135.0,
133.8, 122.2, 117.5, 111.5, 56.6. IR (KBr): 1692, 1492,
1255, 1087, 1019, 761 cmꢀ1. MS m/z: 152 (M+, 100),

S.-T. Huang et al. / Bioorg. Med. Chem. 14 (2006) 6106–6119
6117
4.1.1.31. Compound 32. Yield: 9%. mp: 137–139 ꢁC.
1H NMR (300 MHz, CDCl3, d): 11.28 (s, 1H), 8.16–
8.13 (d, J = 7.8 Hz, 1H), 8.04–8.01 (d, J = 7.8 Hz, 1H),
7.94–7.91 (d, J = 7.9 Hz, 1H), 7.50–7.43 (m, 2H), 7.15–
7.12 (d, J = 8.4 Hz, 1H), 7.07–7.02 (dd, J = 7.9, 7.6 Hz,
1H), 4.07 (s, 3H). 13C NMR (125.7 MHz, CDCl3, d):
164.4, 163.7, 158.7, 148.1, 141.4, 134.0, 127.5, 127.3,
124.7, 123.8, 119.7, 117.4, 115.0, 110.0, 52.5. IR (KBr):
1728, 1631, 1290, 1060, 754 cmꢀ1. MS m/z: 270
(M++1, 12), 269 (M+, 73), 238 (24), 237 (100), 209 (9).
HRMS-EI (m/z): [M]+ calcd for C15H11NO4, 269.0688;
found, 269.0680.
119.4, 117.4, 114.8, 112.0, 111.7, 56.1, 52.5. IR (KBr):
1670, 1548, 1349, 1253, 765 cmꢀ1. MS m/z: 419
(M++1, 13), 418 (M+, 49), 386 (11), 253 (16), 252
(100). HRMS-EI (m/z): [M]+ calcd for C23H18N2O6,
418.1165; found, 418.1167.
4.1.1.34. 20-(2-Methoxy-phenyl)-[2,70]bibenzoxazolyl-
7-carboxylic acid methyl ester (35). A solution of com-
pound 34 (0.14 g, 0.33 mmol) and p-toluenesulfonic acid
(0.16 g, 0.84 mmol) in anhydrous p-xylene (7.5 mL) was
refluxed for 32.5 h, and the reaction was quenched by
adding saturated sodium bicarbonate solution. The
aqueous phase was extracted with ethyl acetate, and
the extract was washed with brine and dried over anhy-
drous magnesium sulfate. After filtration and concentra-
tion, the resulting residue was purified by flash column
chromatography using silica gel as the stationary phase
and using ethyl acetate/hexane (1:9, 3:7, 1:1) as the mo-
bile phase producing compound 35 (40 mg, 0.1 mmol).
Yield: 30%. mp: 164–166 ꢁC. 1H NMR (300 MHz,
CDCl3, d): 8.51–8.49 (d, J = 6.1 Hz, 1H), 8.32–8.29 (d,
J = 8.1 Hz, 1H), 8.09–8.06 (m, 3H), 7.61–7.46 (m, 3H),
7.22–7.13 (m, 2H), 4.12 (s, 3H), 4.09 (s, 3H). 13C
NMR (75 MHz, CDCl3, d): 164.6, 162.4, 160.8, 158.8,
150.8, 149.6, 147.7, 143.8, 143.4, 133.2, 131.4, 127.5,
125.0, 124.9, 124.6, 123.8, 120.7, 115.5, 115.0, 112.1,
111.0, 56.3, 52.4. IR (KBr): 3397 (br), 3079, 3020,
4.1.1.32.
2-(2-Methoxy-phenyl)-benzoxazole-7-car-
boxylic acid (33). To a solution of compound 31
(52 mg, 0.18 mmol) in tetrahydrofuran (3 mL) was add-
ed 5 N sodium hydroxide solution (0.73 mL), and re-
fluxed for 2 days. After cooled to room temperature,
the reaction solution was acidified by concd hydrochlo-
ric acid to give a white precipitate. The precipitate was
filtered and washed with water, and dried under vacuum
to produce compound 33 (45 mg, 0.17 mmol). Yield:
1
91%. mp: 275 ꢁC (decomposed). H NMR (300 MHz,
CD3OD, d): 8.32–8.28 (dd, J = 7.8, 7.8 Hz, 1H), 7.86–
7.83 (d, J = 7.7 Hz, 1H), 7.79–7.76 (d, J = 7.9 Hz, 1H),
7.60–7.54 (dd, J = 7.8, 7.7 Hz,1H), 7.40–7.35 (dd,
J = 7.8, 7.8 Hz, 1H), 7.24–7.21 (d, J = 7.9 Hz, 1H),
7.16–7.10 (m, 1H), 4.0 (s, 3H). 13C NMR (125.7 MHz,
CDCl3, d): 170.7, 161.9, 158.7, 148.4, 142.2, 133.1,
131.0, 126.5, 123.7, 123.2, 120.6, 120.3, 115.1, 111.9,
2916, 2852, 1731, 1617, 1542, 1291, 1431, 746 cmꢀ1
.
MS m/z: 401 (M++1, 26), 154 (35), 81 (39), 69 (60), 57
(74), 55 (100). HRMS-FAB (m/z): [M + H]+ calcd for
C23H16N2O5, 401.1137; found, 401.1142.
54.9. IR (KBr): 3432, 1581, 1400, 1124, 763, 617 cmꢀ1
.
MS m/z: 269 (M+, 4) 175 (100), 154 (50), 137 (25), 136
(34), 57 (22). HRMS-FAB (m/z): [M + H]+ calcd for
C15H11NO4, 270.0766; found, 270.0775.
4.1.1.35. 2-(2-Benzyloxy-benzoylamino)-benzoic acid
methyl ester (36). A solution of compound 10 (2.00 g,
8.8 mmol) in anhydrous dichloromethane (60 mL) was
added oxalyl chloride (3.8 mL, 43.6 mmol), and stirred
for 10 min at room temperature; following added N,N-
dimethylformamide (0.1 mL), and stirred for 2 h. Then
the solvent was removed and dried under vacuum for
3 h. The residue was dissolved in anhydrous dichloro-
methane (50 mL). To this solution was added a solution
of compound 2 (1.32 g, 8.8 mmol) and pyridine (3 mL)
in anhydrous dichloromethane (40 mL) via canula, and
stirred overnight. The reaction was quenched by adding
water and extracted with dichloromethane. The organic
phase was dried over anhydrous magnesium sulfate.
After filtration and concentration, the resulting residue
was purified by flash column chromatography using sil-
ica gel as the stationary phase and using ethyl acetate/
hexane (1:49) as the mobile phase producing compound
4.1.1.33. 2-Hydroxy-3-{[2-(2-methoxy-phenyl)-benz-
oxazole-7-carbonyl]-amino}-benzoic acid methyl ester
(34). A solution of compound 33 (23 mg, 0.09 mmol)
in anhydrous dichloromethane (7 mL) was added oxalyl
chloride (22 lL, 0.26 mmol), and stirred for 10 min at
room temperature; following added 5 drops of N,N-di-
methylformamide, and stirred for 2 h. Then the solvent
was removed and dried under vacuum for 3 h. The res-
idue was dissolved in anhydrous dichloromethane
(10 mL). To this solution was added a solution of com-
pound 29 (18 mg, 0.1 mmol) and pyridine (0.1 mL) in
anhydrous dichloromethane (5 mL) via canula, and stir-
red overnight. The reaction was quenched by adding
water and extracted with dichloromethane. The organic
phase was dried over anhydrous magnesium sulfate.
After filtration and concentration, the resulting residue
was purified by flash column chromatography using sil-
ica gel as the stationary phase and using ethyl acetate/
hexane (1:4, 3:7) as the mobile phase producing com-
pound 34 (30 mg, 0.07 mmol). Yield: 67%. mp: 235–
1
36 (2.55 g, 7.0 mmol). Yield: 81%. mp: 116-119 ꢁC. H
NMR (300 MHz, CDCl3, d) : 12.7 (s, 1H), 8.95–8.92
(d, J = 8.6 Hz, 1H), 8.13–8.11 (d, J = 7.8 Hz, 1H),
8.02–7.99 (d, J = 8 Hz, 1H), 7.45–7.42 (dd, J = 7.3,
6.4 Hz, 1H), 7.45–7.28 (m, 6H), 7.13–7.03 (m, 2H),
7.0–6.97 (d, J = 8.3 Hz, 1H), 5.43 (s, 2H), 3.73 (s, 3H).
13C NMR (125.7 MHz, CDCl3, d): 167.6, 164.7, 156.3,
141.2, 136.7, 134.1, 132.8, 132.1, 130.7, 128.5, 127.7,
126.8, 123.7, 122.5, 121.8, 121.2, 116.5, 113.3, 70.4,
51.9. IR (KBr): 1710, 1586, 1450, 1265, 757 cmꢀ1. MS
m/z: 361 (M+, 5), 241 (16), 240 (19), 151 (29), 92 (14),
91 (100). HRMS-EI (m/z): [M]+ calcd for C22H19NO4,
361.1314; found, 361.1308.
1
238 ꢁC. H NMR (300 MHz, CDCl3, d): 11.61 (s, 1H),
9.89 (s, 1H), 8.90–8.87 (d, J = 7.8 Hz, 1H), 8.43–8.40
(d, J = 7.9 Hz, 1H), 8.22–8.20 (d, J = 7.8 Hz, 1H),
8.03–8.01 (d, J = 7.8 Hz, 1H), 7.66–7.51 (m, 3H), 7.22–
7.12 (m, 2H), 7.03–6.98 (dd, J = 7.8, 7.8 Hz, 1H), 4.05
(s, 3H), 4.01 (s, 3H). 13C NMR (125.7 MHz, CDCl3,
d): 171.0, 161.1, 158.7, 150.8, 146.6, 142.6, 133.5,
131.4, 127.7, 126.7, 125.7, 124.8, 124.3, 124.0, 120.7,

6118
S.-T. Huang et al. / Bioorg. Med. Chem. 14 (2006) 6106–6119
4.1.1.36. 2-(2-Benzyloxy-thiobenzoylamino)-benzoic
adding saturated sodium bicarbonate solution and
extracted with diethyl ether. The extract was dried over
anhydrous magnesium sulfate. After filtration and con-
centration, the residue was purified by flash column chro-
matography using silica gel as the stationary phase and
using ethyl acetate/hexane (1:49, 1:19) as the mobile phase
producing compound 39 (56.0 mg, 0.2 mmol). Yield:
acid methyl ester (37). To a solution of compound 36
(0.36 g, 1.0 mmol) in anhydrous toluene (4 mL) was
added the Lawesson’s reagent (0.40 g, 1.0 mmol) in
glove box. The resulting solution was refluxed for
7.5 h under argon atmosphere, and the reaction was
quenched by adding water. The aqueous solution was
extracted with ethyl acetate, and the extract was dried
over anhydrous magnesium sulfate. After filtration and
concentration, the residue was purified by flash column
chromatography using silica gel as the stationary phase
and using ethyl acetate/hexane (1:49) as the mobile
phase producing compound 37 (0.19 g, 0.5 mmol).
Yield: 51%. mp: 74–76 ꢁC. 1H NMR (300 MHz, CDCl3,
d): 12.71 (b, 1H), 9.59–9.56 (d, J = 8.2 Hz, 1H), 8.04–
8.01 (d, J = 7.6 Hz, 1H), 7.96–7.94 (d, J = 7.2 Hz, 1H),
7.65–7.60 (dd, J = 7.6, 6.8 Hz, 1H), 7.37–7.32 (m, 3H),
7.23–7.21 (m, 4H), 7.06–6.98 (m, 2H), 5.20 (s, 2H),
3.79 (s, 3H). 13C NMR (125.7 MHz, CDCl3, d): 134.0,
133.7, 133.6, 132.3, 131.5, 130.9, 128.6, 128.4, 127.7,
126.6, 124.9, 121.9, 121.3, 118.4, 113.3, 70.6, 52.3. IR
(KBr): 1703, 1588, 1449, 1349, 1267, 759 cmꢀ1. MS m/
z: 377 (M+, 0.61), 345 (25), 344 (100), 318 (20), 312
(27). HRMS-EI (m/z): [M]+ calcd for C22H19NO3S,
377.1086; found, 377.1091.
1
81%. mp: 163–165 ꢁC. H NMR (300 MHz, CDCl3, d):
8.24–8.21 (d, J = 7.7 Hz, 1H), 8.12–8.09 (d, J = 8.0 Hz,
1 H), 7.71–7.68 (d, J = 7.9 Hz, 1H), 7.52–7.47 (dd,
J = 7.9, 7.8 Hz, 1H), 7.45–7.40 (dd, J = 8.0, 7.9 Hz, 1H),
7.18–7.16 (d, J = 8.4 Hz, 1H), 7.00-6.94 (t, J = 8.0,
6.4 Hz, 1H), 4.09 (s, 3H). 13C NMR (125.7 MHz, CDCl3,
d): 170.7, 165.9, 158.7, 149.4, 134.1, 133.5, 129.7, 128.4,
126.0, 125.0, 123.1, 119.4, 118.2, 116.3, 52.5. IR (KBr):
1717, 1487, 1258, 1114, 741 cmꢀ1. MS m/z: 286 (M++1,
9), 285 (M+, 59), 254 (18), 253 (100), 225 (10). HRMS-
EI (m/z): [M]+ calcd for C15H11NO3S, 285.0460; found,
285.0466.
4.1.1.39. 2-(2-Benzyloxy-phenyl)-benzothiazole-4-car-
boxylic acid (40). To a solution of compound 38 (0.21 g,
0.6 mmol) in tetrahydrofuran (10.5 mL) was added 1 N
sodium hydroxide solution (16.1 mL), and refluxed over-
night. After cooled to room temperature, the reaction
solution was acidified by concd hydrochloric acid to give
a white precipitate. The precipitate was filtered and
washed with water, and dried under vacuum to produce
compound 40 (0.19 g, 0.5 mmol). Yield: 95%. mp: 223–
4.1.1.37. 2-(2-Benzyloxy-phenyl)-benzothiazole-4-car-
boxylic acid methyl ester (38). To a solution of potassi-
um ferricyanide(III) (2.87 g, 8.7 mmol) in water (2 mL)
at 80–90 ꢁC was added a solution of compound 37
(0.82 g, 2.2 mmol) in ethanol (1 mL), 30 % sodium
hydroxide solution (1.68 mL), and water (0.72 mL) at
a rate of 5 mL per minute. After stirred for 1 h, the reac-
tion solution was cooled to room temperature and
poured into water. The resulting solution was extracted
with ethyl acetate, and the extract was dried over anhy-
drous magnesium sulfate. After filtration and concentra-
tion, the residue was dissolved in a mixture of diethyl
ether and methanol, and added diazomethane at 0 ꢁC
until ceased to generate nitrogen. After adding a few
drops of acetic acid, the reaction solution was concen-
trated, and the residue was purified by flash column
chromatography using silica gel as the stationary phase
and using hexane and then ethyl acetate/hexane (1:49) as
the mobile phase producing compound 38 (0.17 g,
0.5 mmol). Yield: 21%. mp: 112–116 ꢁC. 1H NMR
(300 MHz, CDCl3, d): 8.73–8.70 (d, J = 7.9 Hz, 1H),
8.11–8.04 (m, 2H), 7.54–7.52 (d, J = 7.6 Hz, 2H), 7.45–
7.32 (m, 5H), 7.17–7.09 (m, 2H), 5.36 (s, 2H), 4.07 (s,
3H). 13C NMR (125.7 MHz, CDCl3, d): 166.9, 164.8,
156.4, 149.9, 137.8, 135.9, 132.2, 130.3, 128.68, 128.66,
128.3, 127.8, 125.7, 124.3, 123.7, 122.3, 121.4, 112.7,
71.0, 52.4. IR (KBr): 1696, 1452, 1292, 1241, 1113,
751 cmꢀ1. MS m/z: 376 (M++1, 14), 375 (M+, 54), 358
(20), 269 (12), 211 (11). HRMS-EI (m/z): [M]+ calcd
for C22H17NO3S, 375.0929; found, 375.0925.
1
225 ꢁC. H NMR (300 MHz, CDCl3, d)g 8.47–8.43 (d,
J = 7.9 Hz, 1H), 8.38–8.35 (d, J = 8.0 Hz, 1H), 8.09–
8.06 (d, J = 8.0 Hz, 1H), 7.55–7.50 (m, 4H), 7.47–7.41
(m, 3H), 7.22–7.16 (m, 2H), 5.39 (s, 2H). 13C NMR
(125.7 MHz, CDCl3, d): 166.0, 156.7, 149.6, 135.8,
135.4, 133.4, 129.6, 129.5, 128.7, 128.6, 128.0, 126.3,
125.1, 121.7, 121.6, 120.5, 113.0, 71.2. IR (KBr): 1731,
1454, 1248, 994, 747 cmꢀ1. MS m/z: 362 (M++1, 12), 361
(M+, 53), 317 (15), 253 (16), 211 (10). HRMS-EI (m/z):
[M]+ calcd for C21H15NO3S, 361.0773; found, 361.0780.
4.1.1.40. 2-{[2-(2-Benzyloxy-phenyl)-benzothiazole-4-
carbonyl]-amino}-benzoic acid methyl ester (41). A solu-
tion of compound 40 (0.10 g, 0.3 mmol) in anhydrous
dichloromethane (4 mL) was added oxalyl chloride
(1.0 mL, 11.5 mmol), and stirred for 10 minutes at room
temperature; following added 5 drops of N,N-dimethyl-
formamide, and stirred for 2 h. Then the solvent was re-
moved and dried under vacuum for 3 h. The residue was
dissolved in anhydrous dichloromethane (2 mL). To this
solution was added a solution of compound 2 (0.05 g,
0.3 mmol) and pyridine (0.5 mL) in anhydrous dichloro-
methane (3 mL) via canula, and stirred overnight. The
reaction was quenched by adding water and extracted
with dichloromethane. The organic phase was dried over
anhydrous magnesium sulfate. After filtration and con-
centration, the resulting residue was purified by flash col-
umn chromatography using silica gel as the stationary
phase and using ethyl acetate/hexane (1:49) as the mobile
phase producing compound 41 (0.08 g, 0.2 mmol). Yield:
4.1.1.38. 2-(2-Hydroxy-phenyl)-benzothiazole-4-car-
boxylic acid methyl ester (39). To a solution of compound
38 (91.0 mg, 0.2 mmol) in anhydrous dichloromethane
(2 mL) were added ethanethiol (1.5 mL) and boron triflu-
oride diethyl etherate (1.0 mL), and stirred at room tem-
perature for 5 days. The reaction was quenched by
1
54%. mp: 233–235 ꢁC. H NMR (300 MHz, CDCl3, d):
12.9 (s, 1H), 8.73–8.70 (d, J = 7.6 Hz, 1H), 8.65–8.62 (d,
J = 7.6 Hz, 1H), 8.47–8.45 (d, J = 7.6 Hz, 1H), 8.12–
8.09 (d, J = 8.0 Hz, 1H), 8.06–8.03 (d, J = 7.9 Hz, 1H),

S.-T. Huang et al. / Bioorg. Med. Chem. 14 (2006) 6106–6119
6119
7.57–7.42 (m, 8H), 7.24–7.18 (dd, J = 7.6, 7.6 Hz, 1H),
7.14–7.05 (m, 2H), 5.38 (s, 2H), 3.74 (s, 3H). 13C NMR
(125.7 MHz, CDCl3, d): 167.2, 164.4, 164.1, 156.6,
148.8, 140.1, 136.9, 135.8, 133.6, 132.3, 131.0, 130.5,
128.8, 128.7, 128.4, 127.9, 126.7, 125.0, 124.5, 124.1,
123.4, 121.9, 121.1, 119.2, 112.7, 71.1, 52.1. IR (KBr):
1718, 1662, 1535, 1449, 1244, 752 cmꢀ1. MS m/z: 495
(M++1, 33), 494 (M+, 100), 344 (30), 254 (37), 253 (94).
HRMS-EI (m/z): [M]+ calcd for C29H22N2O4S,
494.1300; found, 494.1304.
rate) · 100%. Therefore, the negative control was set
to 100% survival or 0% toxicity.
Acknowledgments
This paper is dedicated to Professor Yoshito Kishi on
the occasion of his 70th birthday. The authors thank
Ms. L. M. Hsu, at Instruments Center, National Chung
Hsing University, and Ms. H. C. Tan, at Instrument
Center, National Tsing Hwa University, for their help
in obtaining HRMS and 13C NMR spectra, and the
National Science Council the Republic of China for
financially supporting this research under contract
NSC 93-2323-B-259-002.
4.2. Biological evaluation
4.2.1. Cell culture. The human bladder papillary transi-
tional cell carcinoma cell line BFTC-905, the human lung
carcinoma cell line A-549, the human uterus sarcoma cell
line MES-SA, and the human embryonal rhabdomyosar-
coma cell line RD were obtained from the Bioresources
Collection & Research Center (BCRC, Taiwan). All can-
cer cell lines were maintained in the BCRC recommended
medium supplemented with 10% fetal bovine serum and
penicillin–streptomycin. After 24 h incubation at 37 ꢁC
in humidified 5% carbon dioxide to allow cell attachment,
the cellsweretreatedwith various concentrations ofUK-1
derivatives and incubated for 72 h under the same condi-
tions. Even though the UK-1 derivatives were dissolved in
DMSO, in the treatment stage the final DMSO concentra-
tion never exceeded 0.5%.
References and notes
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18. The steric energy was calculated using the MM2 program
(CS Chem3D Pro).
4.2.2. Cytotoxicity assay. Cell viability was determined
by measuring the metabolism of a tetrazolium substrate,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT).16 Briefly, the cells (1 · 104 cells per
well) were incubated in 96-well plates in which each cell
contained 200 ll of the growth medium at 37 ꢁC. Fol-
lowing overnight incubation, the growth medium was
removed from cultures and replaced with 200 ll of the
test article solution. The MTT test was conducted using
six concentrations, 100 lM, 75 lM, 50 lM, 25 lM,
5 lM, and 0 lM, in four cell lines. Tests at each concen-
tration were conducted in triplicate. Test article solu-
tions were incubated with target cells for
approximately 72 h at 37 1 ꢁC with 5 1% CO2. Fol-
lowing exposure, the test solutions were removed and re-
placed with 500 lg/ml MTT solution. After around 6 h
of incubation, the MTT solution was removed. Sodium
dodecyl sulfate (SDS) solution (10%) was added into cell
cultures and the optical density (OD) values were read
using an ELISA reader (SUNRISE, TECAN, Switzer-
land) at a wavelength of 570 nm. The mean and stan-
dard deviation of each group were calculated. The OD
reading of every group was first substrate with blank
control (background). The relative survival rate was
the OD reading of the treated group divided by that
of the negative control group, multiplied by 100. The
percentage of toxicity was calculated as (1 ꢀ survival